Report
The SET-2/SET1 Histone H3K4 Methyltransferase Maintains Pluripotency in the Caenorhabditis elegans Germline Graphical Abstract
Authors Vale´rie J. Robert, Marine G. Mercier, ..., Rafal Ciosk, Francesca Palladino
Correspondence
[email protected]
In Brief Germ cells are totipotent, meaning that they can give rise to all cells of an organism. Robert et al. identify a function for H3K4 methylation, a histone modification mediated by the SET-2/SET1 methyltransferase, in repressing somatic gene expression in the C. elegans germline. In the absence of SET-2/SET1 and H3K4 methylation, germ cells differentiate into somatic cells, indicating that H3K4 methylation helps preserve germ cell identity.
Highlights
Accession Numbers
H3K4 methylation represses expression of somatic genes in the C. elegans germline
GSE61094
Loss of H3K4 methylation results in germline transdifferentiation Transdifferentiation increases across generations The nuclear RNAi pathway contributes to the maintenance of germ cell identity
Robert et al., 2014, Cell Reports 9, 443–450 October 23, 2014 ª2014 The Authors http://dx.doi.org/10.1016/j.celrep.2014.09.018
Cell Reports
Report The SET-2/SET1 Histone H3K4 Methyltransferase Maintains Pluripotency in the Caenorhabditis elegans Germline Vale´rie J. Robert,1,3 Marine G. Mercier,1,3 Ce´cile Bedet,1 Ste´phane Janczarski,1 Jorge Merlet,2 Steve Garvis,1 Rafal Ciosk,2 and Francesca Palladino1,* 1Laboratory
of Molecular and Cellular Biology, CNRS, Universite´ de Lyon 1, Ecole Normale Supe´rieure, 69364 Lyon Cedex 07, France Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland 3Co-first author *Correspondence:
[email protected] http://dx.doi.org/10.1016/j.celrep.2014.09.018 This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/). 2Friedrich
SUMMARY
Histone H3 Lys 4 methylation (H3K4me) is deposited by the conserved SET1/MLL methyltransferases acting in multiprotein complexes, including Ash2 and Wdr5. Although individual subunits contribute to complex activity, how they influence gene expression in specific tissues remains largely unknown. In Caenorhabditis elegans, SET-2/SET1, WDR-5.1, and ASH-2 are differentially required for germline H3K4 methylation. Using expression profiling on germlines from animals lacking set-2, ash-2, or wdr-5.1, we show that these subunits play unique as well as redundant functions in order to promote expression of germline genes and repress somatic genes. Furthermore, we show that in set-2- and wdr-5.1-deficient germlines, somatic gene misexpression is associated with conversion of germ cells into somatic cells and that nuclear RNAi acts in parallel with SET-2 and WDR-5.1 to maintain germline identity. These findings uncover a unique role for SET-2 and WDR-5.1 in preserving germline pluripotency and underline the complexity of the cellular network regulating this process. INTRODUCTION Methylation of H3K4 at promoters of actively transcribed genes is catalyzed by the conserved class of SET1/MLL histone methyltransferases (HMTs) acting in multiprotein complexes that share common subunits, including WDR5 and ASH2 (Eissenberg and Shilatifard, 2010). While in yeast SET1 is solely responsible for all H3K4 methylation, in mammalian cells SET1A and SET1B and four mixed-lineage leukemia (MLL)-family HMTs (MLL1–4) have been characterized, suggesting complex regulation in higher eukaryotes. In addition, ASH2, WDR5, and RBBP5 have been identified in complexes in the absence of SET1- or MLLrelated proteins, further adding to this complexity (Dehe´ et al., 2006; Patel et al., 2009; Steward et al., 2006).
H3K4 can be mono- (me1), di- (me2), or trimethylated (me3), with H3K4me3 enrichment found near active promoters and H3K4me2/1 found at both promoter and enhancer regions (Bernstein et al., 2005; Heintzman et al., 2007). While SET1A/SET1B catalyze the majority of H3K4me3, MLL1/2 maintain H3K4me3 at discrete genomic sites and MLL3/4 catalyze H3K4me1 (Bhagwat and Vakoc, 2014). How H3K4 methylation contributes to transcription is poorly understood. H3K4me3 correlates with transcriptional activation and is coupled to the activities of other chromatin-modifying enzymes (Eissenberg and Shilatifard, 2010). However, impairment of H3K4 methylation results in minimal transcriptional effects (Jiang et al., 2011; Lenstra et al., 2011), and transcriptional activation can occur in its absence (Ho¨dl and Basler, 2012). More recently, it has been shown that in yeast, H3K4 methylation can also repress gene expression through antisense transcription (Margaritis et al., 2012; Pinskaya and Morillon, 2009). Dynamic changes in H3K4me2 and H3K4me3 are observed during cellular reprogramming, both as part of a global early response in the absence of transcriptional activation as well as on activated pluripotency-associated targets (Gaspar-Maia et al., 2011; Koche et al., 2011). H3K4me3 methylation is also part of a bivalent chromatin mark that typifies poised developmental genes in embryonic stem cells (Voigt et al., 2013). Altogether, these data suggest that direct transcriptional regulation may not be the primary function of H3K4 methylation. C. elegans contains one SET1 protein (SET-2), one MLL-like protein (SET-16), and a single homolog of ASH2. Of the three WDR5 homologs, only WDR-5.1, most closely related to human WDR5, is required for H3K4 methylation (Li and Kelly, 2011; Xiao et al., 2011). We and others have previously shown that SET-2 and ASH-2 are differentially required for global H3K4 methylation in embryos and adult germ cells (Li and Kelly, 2011; Xiao et al., 2011), consistent with diverse H3K4 HMT complexes and methylation states playing distinct roles during development (Bledau et al., 2014; Eissenberg and Shilatifard, 2010). Both set-2 and wrd-5.1 are required for the maintenance of the germline stem cell population, and their absence results in a temperature-sensitive, progressive sterility over generations (Li and Kelly, 2011; Simonet et al., 2007; Xiao et al., 2011), a phenotype not associated with ash-2 loss of function. In this paper, we Cell Reports 9, 443–450, October 23, 2014 ª2014 The Authors 443
sought to gain insight into how SET-2, WDR-5.1, and ASH-2 contribute to germline maintenance through expression profiling of dissected germlines from animals lacking set-2, wdr-5.1, or ash-2. Our data are consistent with these three subunits cooperating to promote gene expression but also having additional unique regulatory functions. Most importantly, our results show that set-2, wdr-5.1, and ash-2 are required to repress expression of somatic genes in the germline. Ectopic expression of somatic genes in set-2 and wdr-5.1 mutant germlines is associated with conversion of germ cells into neuronal and muscle cells. We further show that the nuclear RNAi pathway acts in parallel to SET-2 and WDR-5.1 to maintain germline identity. Our findings identify SET-2, WDR-5.1, and H3K4 methylation as important regulatory elements in maintaining expression of germline genes, repressing somatic genes, and preserving germline pluripotency. RESULTS SET-2, WDR-5.1, and ASH-2 Differentially Contribute to Gene Expression in the C. elegans Germline Transcriptomic analysis was carried out on dissected gonads from fertile set-2, wdr-5.1, and ash-2 loss-of-function (lf) mutants grown at 20 C, focusing our analysis on genes regulated at least 2-fold (p % 0.05; Figures 1A and S1; Table S1). In all three mutant backgrounds, significant numbers of down- and upregulated targets were found. For set-2 and wdr-5.1 germlines, 87% (444/509) and 73% (461/628) of misregulated genes, respectively, were upregulated, consistent with a predominant role in gene repression. By contrast, in ash-2 germlines, approximately the same number of genes was up- and downregulated (136 and 167, respectively). set-2-upregulated genes showed an 8% overlap with wdr-5.1 and ash-2 upregulated genes, whereas downregulated genes showed a 30% overlap with wdr-5.1 and ash-2 downregulated genes (Figure 1B; Table S2). Therefore, SET-2, WDR-5.1, and ASH-2 have a common role in regulating the expression of a subset of target genes. However, the high percentage of remaining, nonoverlapping genes (92% and 70%, respectively), suggests that these subunits also play significant regulatory roles independently of one another. Pairwise comparison of upregulated genes further showed that 44% of set-2 target genes overlap with wdr-5.1, while only 14% overlap with ash-2 targets. Therefore, SET-2 appears to play a repressive role in common with WDR-5.1 and mostly independently of ASH-2. SET-2 and WDR-5.1 Act in the Germline to Promote Expression of Genes Associated with Germline Function and Repress Expression of Somatic Genes Next, we sorted misregulated genes into ‘‘ubiquitous,’’ ‘‘germline-expressed,’’ and ‘‘germline-specific’’ categories according to their expression pattern in serial analysis of gene expression (SAGE) studies (Meissner et al., 2009; Wang et al., 2009) (Figures 1C and 1D). For downregulated targets, germline-expressed genes were significantly overrepresented in the wdr-5.1 data set (p = 1 3 103), and germline-specific genes overrepresented in both set-2 and wdr-5.1 data sets (p = 3 3 105 and 3.3 3 1014, respectively). Therefore, SET-2 and WDR-5.1 promote 444 Cell Reports 9, 443–450, October 23, 2014 ª2014 The Authors
the expression of a small number of germline-expressed genes (Table S3). For genes upregulated in set-2, wdr-5.1, and ash-2 mutant germlines, both ubiquitous and germline-expressed genes were underrepresented (Figure 1D). Further comparison with an assembled list of 3,986 ‘‘soma-specific’’ genes represented by at least three SAGE tags in muscle, neurons, or intestine, but absent from dissected gonads, showed that a majority of genes upregulated in set-2, wdr-5.1, and ash-2 mutant germlines are expressed in somatic tissue (Figures 1E and S1D). Gene ontology (GO) analysis (Figure S1E) further showed that genes related to neuronal function were significantly overrepresented in the set-2 and wdr-5.1 lists, while for genes commonly regulated in the absence of any two subunits, or of the three subunits together, no enrichment in any specific GO term was observed. Altogether, these results point to a requirement for SET-2, WDR-5.1, and ASH-2 in preventing ectopic expression of somatic genes in the C. elegans germline. Somatic Cell Fate Conversion in the Absence of H3K4 Methylation Loss of germline H3K4 methylation in both set-2(lf) and wdr5.1(lf) animals is associated with a temperature-sensitive progressive sterility, known as the mortal germline (Mrt) phenotype (Li and Kelly, 2011; Simonet et al., 2007; Xiao et al., 2011). In the gonad of set-2(lf) animals nearing sterility at 25 C, many nuclei lost the typical ‘‘fried-egg shape’’ and were instead smaller with a granular nucleoplasm, resembling neuronal cells (Figure 2A) (Ciosk et al., 2006). Antibody staining showed that germline-specific P granules (Strome and Wood, 1982), which are present in wild-type and fertile set-2 animals at 20 C, are lost in late-generation set-2 mutant germlines at 25 C (Figures 2B and S2A). To further investigate somatic conversion of germ cells lacking set-2, we used an unc-119::GFP transgenic reporter uniquely expressed in multiple neurons in wild-type animals (Ciosk et al., 2006) and antibodies specifically recognizing muscle cells. We observed that a subset of the abnormal nuclei in set-2 mutant germlines expressed the unc-119::GFP reporter, the body wall muscle marker UNC-120 (a MADS box factor), or myosin (Figures 2C, 2D, and S2B). unc-119::GFP expression was often observed in cells with extensions resembling axodendritic projections (Figure 2C). Only 18% (4/22) of germlines expressing neuronal GFP also expressed UNC-120, while all germlines expressing UNC-120 were also positive for neuronal GFP. By contrast, ELT-2, a marker of intestinal differentiation, was not observed in either neuronal GFP-expressing (n = 30) or nonexpressing (n = 32; Figure S2C) germlines. Altogether, these results show that set-2(lf) germlines have acquired somatic cell fate and suggest that conversion into neuronal cell fate may be more easily achieved than other somatic fates (Patel et al., 2012). To ask whether germ cell conversion in set-2 and wdr-5.1 mutants correlates with loss of germ cell immortality, we carried out transgenerational analysis of unc-119::GFP germline expression at 25 C. In early generations (F1 or F2, Table 1), when set-2(lf) animals are still fertile, most germlines did not express GFP (Figure S2D). This number strongly increased in the F3 generation, at the onset of sterility. Similar result were obtained in wdr5.1(lf) animals, but not set-2(ok952) animals, in which H3K4
A
set-2 vs WT
wdr-5.1 vs WT
83 probes 65 genes
15
608 probes 444 genes
- log10 p value
- log10 p value
15
10
5
175 probes 167 genes
607 probes 461 genes
10
5
0
0 - 10
-5
0
5
10
- 10
-5
log2 Fold Change
- log10 p value
5
10
B
ash-2 vs WT 15
0
log2 Fold Change
175 probes 136 genes
set-2 (65)
227 probes 167 genes
ash-2 (136)
set-2 (444)
ash-2 (167)
10
5
wdr-5.1 (461)
wdr-5.1 (167)
0 - 10
-5
0
5
Downregulated
10
Upregulated
log2 Fold Change
15
ns ns
3e-5
ns
40 30
0
se t-2 wd r-5 .1 as h-2
0
se t-2 wd r-5 .1 as h-2
10
Ubiquitous genes (2341)
Germline Germline specific expressed genes (2857) genes (516)
2e-7
20
5
3.5e-9 2e-3 ns ns
Germline Ubiquitous expressed genes (2341) genes (2857)
Germline specific genes (516)
% of upregulated targets
ns 1e-6
observed expected
100 80 60 40 20 0 se t wd -2 r-5 as .1 h2
ns
20
60 1e-12
